The use of bacterial cells to produce protein based therapeutics is increasing in commercial importance. One of the goals in developing a bacterial expression system is the production of high quality target polypeptides quickly, efficiently, and abundantly. An ideal host cell for such an expression system would be able to efficiently utilize a carbon source for the production of a target polypeptide, quickly grow to high cell densities in a fermentation reaction, express the target polypeptide only when induced, and grow on a medium that is devoid of regulatory and environmental concerns.
There are many hurdles to the creation of a superior host cell. First, in order to produce a recombinant polypeptide, an expression vector encoding the target protein must be inserted into the host cell. Many bacteria are capable of reverting back into an untransformed state, wherein the expression vector is eliminated from the host. Such revertants can decrease the fermentation efficiency of the production of the desired recombinant polypeptide.
Expression vectors encoding a target peptide typically include a selection marker in the vector. Often, the selection marker is a gene whose product is required for survival during the fermentation process. Host cells lacking the selection marker, such as revertants, are unable to survive. The use of selection markers during the fermentation process is intended to ensure that only bacteria containing the expression vector survive, eliminating competition between the revertants and transformants and reducing the efficiency of fermentation.
The most commonly used selection markers are antibiotic resistance genes. Host cells are grown in a medium supplemented with an antibiotic capable of being degraded by the selected antibiotic resistance gene product. Cells that do not contain the expression vector with the antibiotic resistance gene are killed by the antibiotic. Typical antibiotic resistance genes include tetracycline, neomycin, kanamycin, and ampicillin. The presence of antibiotic resistance genes in a bacterial host cell, however, presents environmental, regulatory, and commercial problems. For example, antibiotic resistance gene-containing products (and products produced by the use of antibiotic resistance gene) have been identified as potential biosafety risks for environmental, human, and animal health. For example, see M. Droge et al., Horizontal Gene Transfer as a Biosafety issue: A natural phenomenon of public concern, J. Biotechnology. 64(1): 75-90 (17 Sep. 1998); Gallagher, D. M., and D. P. Sinn. 1983. Penicillin-induced anaphylaxis in a patient under hypotensive anaesthesia. Oral Surg. Oral Med. Oral Pathol. 56:361-364; Jorro, G., C. Morales, J. V. Braso, and A. Pelaez. 1996. Anaphylaxis to erythromycin. Ann. Allergy Asthma Immunol. 77:456-458; F. Gebhard & K. Smalla, Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA, Appl. & Environ. Microbiol. 64(4):1550-54 (April 1998); T. Hoffmann et al., Foreign DNA sequences are received by a wild type strain of Aspergillus niger after co-culture with transgenic higher plants, Curr. Genet. 27(1): 70-76 (December 1994); DK Mercer et al., Fate of free DNA and transformation of the oral bacterium Streptococcus gordonoii DL1 by plasmid DNA in human saliva, Appl. & Environ. Microbiol. 65(1):6-10 (January 1999); R. Schubbert et al., Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA, PNAS USA 94:961-66 (Feb. 4, 1997); and AA Salyers, Gene transfer in the mammalian intestinal tract, Curr. Opin. in Biotechnol. 4(3):294-98 (June 1993).
As a result of these concerns, many governmental food, drug, health, and environmental regulatory agencies, as well as many end users, require that antibiotic resistance gene nucleic acid be removed from products or be absent from organisms for use in commerce. In addition, evidence demonstrating clearance of the selection antibiotics from the final product must be provided in order to secure regulatory clearance. The United Kingdom, Canada, France, the European Community, and the United States have all addressed the use of antibiotic resistance genes in foods, animal feeds, drugs and drug production, including recombinant drug production. Clearance of these agents, and especially demonstrating such clearance, is expensive, time consuming, and often only minimally effective.
Because of the concerns inherent in the use of antibiotic resistance genes for selection in the production of recombinant polypeptides, alternative selection methods have been examined.
Auxotrophic Selection Markers
Auxotrophic selection markers have been utilized as an alternative to antibiotic selection in some systems. For example, auxotrophic markers have been widely utilized in yeast, due largely to the inefficiency of antibiotic resistance selection markers in these host cells. See, for example, J T Pronk, (2002) “Auxotrophic yeast strains in fundamental and applied research,” App. & Envirn. Micro. 68(5): 2095-2100; Boeke et al., (1984) “A positive selection for mutants lacking orotodine-5′-phosphate decarboxylase activity in yeast; 5-fluoro-orotic acid resistance,” Mol. Gen. Genet. 197: 345-346; Botstein & Davis, (1982) “Principles and practice of recombinant DNA research with yeast,” p. 607-636, in J N Strathern, E W Jones. And J R Broach (ed.), The molecular biology of the yeast Saccharomyces cerevisiae, Metabolism and gene expression, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Cost & Boeke, (1996) “A useful colony color phenotype associated with the yeast selectable/counter selectable marker MET15,” Yeast 12: 939-941. However, yeast expression systems due not provide the potential speed and efficiency for producing target proteins that bacterial systems do.
Auxotrophic marker selection in bacteria has also previously been described. See, for example, U.S. Pat. Nos. 4,920,048, 5,691,185, 6,291,245, 6,413,768, 6,752,994, Struhl et al. (1976) PNAS USA 73; 1471-1475; MacCormick, C. A., et al., (1995) “Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon,” FEMS Microbiol. Lett. 127:105-109; Dickely et al. (1995), “Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector,” Mol. Microbiol. 15:839-847; Sørensen et al., (2000) “A food-grade cloning system for industrial strains of Lactococcus lactis,” Appl. Environ. Microbiol. 66:1253-1258; Fiedler & Skerra, (2001) “proBA complementation of an auxotrophic E. coli strain improves plasmid stability and expression yield during fermenter production of a recombinant antibody fragment,” Gene 274: 111-118.
The use of auxotrophic selection markers in the previously described commercial scale bacterial fermentation systems has drawbacks that limit their use. A major drawback, as noted in U.S. Pat. No. 6,413,768, is that nutritional auxotrophic selection marker systems generally suffer from cross feeding. The term cross feeding refers to the ability of a first cell, auxotrophic for a particular metabolite, to survive in the absence of the metabolite by obtaining its supply of that metabolite from its environment, and typically, from the medium for which the cell is auxotrophic by utilizing excreted intermediates of the metabolite, the metabolite itself, or a prototrophic enabling molecule produced by a second cell, prototrophic for the metabolite absent from the medium. See also GR Barker et al., Biochem. J. 157(1):221-27 (1976) (cross feeding of thymine in E. coli): T J Kerr & G J Tritz, J. Bact. 115(3):982-86 (September 1973) (cross feeding of NAD in E. coli auxotrophic for NAD synthesis); G A Sprenger et al., FEMS Microbiol. Lett. 37(3):299-304 (1986) (selection of nalidixic acid to avoid the cross feeding problem).
Because cross feeding allows revertant bacteria to survive, cross feeding decreases the overall capacity of the fermentation process to produce the desired product at efficient and maximized levels due to the presence of fewer target protein producing host cells.
Expression Vector Control
Another hurdle to the creation of the ideal host cell is the inefficient and low level production of target polypeptides in the fermentation process. Controlling expression of the target protein until optimal host cell densities and fermentation conditions are reached allows for a more efficient and larger yield of polypeptide. The reasons for this are several fold, including a more efficient utilization of a particular carbon source and the reduction of extended metabolic stresses on the host cell.
In many cases, however, repression of expression of the target protein during cell growth can be imperfect, resulting in a significant amount of expression prior to the particular induction phase. This “leaky” repression results in host cell stress, inefficient utilization of carbon source due to metabolic energy being diverted from normal cell growth to transgene, and a delay in reaching optimal cell density induction points, resulting in a more lengthy and costly fermentation run, and often, a reduced yield of the target protein.
Therefore, it is an object of the present invention to provide an improved expression system for the production of target proteins, wherein the production is efficient, regulatable, and performed in a medium that minimizes of regulatory and environmental concerns.
It is another object of the present invention to provide organisms for use as host cells in an improved expression system for the production of target proteins.
It is still another object of the present invention to provide processes for the improved production of target proteins.
It is yet another object of the present invention to provide novel constructs and nucleic acids for use in an improved expression system for the production of target proteins.